**5. Starch hydrolysis with diluted phosphoric acid**

some Brazilian partners in this project applied steam explosion to the pretreatment of sugarcane biomass with almost exactly the same kinetic conditions of our oPA treatment [48]., omitting, unfortunately and consciously, to mention our pioneering publication, as it is completely

*"Steam explosion of cane bagasse using phosphoric acid catalysis", IBS2010 – 14th Intl. Biotechnology* 

bagasse for cattle feeding [51]. Enhanced ruminal degradability (almost 70%) was obtained by adding 2.9% (w/w) in comparison to 60% achieved with solvolysis with water (197°C,13.5 atm,

not have to be washed out prior to fermentation because phosphate can act as an important micronutrient, after partial neutralization with ammonia, for the subsequent fermentation

Steam treatment of sugarcane bagasse with a low level of phosphoric acid (1% of bagasse dry weight) at elevated temperatures (160–190°C) during 10 min resulted in a total sugar yield ranging from 215 to 299 g/kg bagasse (untreated dry weight) and lower levels of products from sugar degradation (furans and organic acids) in all treatment temperatures (140–190°C) as compared to sulfuric acid [53]. Hemicellulose hydrolysates from treatment temperatures below 180°C could be fermented (slowly) by ethanologenic *E. coli* without the need of purifi-

In another study, hemicelluloses from sugarcane bagasse were efficiently solubilized (96% and 98% after 8 and 24 min, respectively) using a low concentration of phosphoric acid (0.20%) at 186°C [54]. Enzymatic cellulose conversion of pretreated bagasse using 20 filter paper cellulase units (FPU) g−1 of Novozymes Celluclast® (a commercial cellulase preparation produced by a selected strain of the fungus *Trichoderma reesei*) treated under these conditions of pretreatment produced the highest cellulose conversion of 56.38%. In general low levels of degradation products were achieved; however, minor increase of these products were observed when temperature was elevated to 186°C that can be explained by the high

Mild phosphoric pretreatment has been also adopted with stream treated substrates. Preimpregnation of *Eucalyptus benthamii* with diluted phosphoric acid followed by steam explosion resulted in an improved selectivity towards hemicellulose hydrolysis (xylose yields of 50–60%), yielding substrates readily susceptible to saccharification with Novozymes Cellic® CTec2 (a commercial enzymatic blend to produce cellulosic ethanol) at relatively high solids

Results obtained on sugarcane bagasse through a central composite design comparing steam explosion carried out in the absence (autohydrolysis) and presence of phosphoric acid showed that phosphoric acid catalysis (19 mg g−1) resulted in better glucan yields under milder conditions (180°C, 5 min) [56]. Phosphoric acid catalysis produced steam-treated substrates with good susceptibility to enzymatic hydrolysis (30 mg g−1 Cellic® CTec2, at 8% of substrate con-

.

was used to increase the nutritional value of sugar cane

generates less carbohydrate dehydration and does

*Symposium and Exhibition, Palacongressi, Rimini, Italy; 14–18 Sept, 2010."*

PO4

PO4

cation [53]. This demonstrated low level of potential inhibitors.

solubilization of hemicellulose fraction at this condition [54]

sistency) yielding in average 75% of glucose.

clear from their below report at Italy.

252 Sugarcane - Technology and Research

In our another work, aqueous H3

4:1 w/w of water). Furthermore, H3

step [51, 52].

(10%) [55].

Starch is the second most abundant polymer in the world [59]. Starch granules are biosynthesized reserve polysaccharide in a broad array of plant tissues and within many plant species. Potatoes and cassava are outstanding starch sources. They are composed of two types of *α*-linked glucans: amylose, a straight chain of *α*-1,4-linked glucopyranosyl units and amylopectin, which has besides *α*-1,4-linked glucopyranosyl units various branch points with *α*-1,6-linkages. A linear polymer of amylose (around 20% of whole starch) can have up to 6000 glucose units, whereas amylopectin (around 80% of the whole starch) is composed of *α*-1,4-linked chains of 10–60 glucose units with *α*-1,6-linked side chains of 15–45 glucose units. Both building blocks represent approximately 98–99% of the starch dry weight [60].

Starch may be chemically, enzymatically or physically modified to produce a broth rich in glucose that possess potential use in biotechnological processes, such as fermentation substrate for microorganisms to produce bioethanol, enzymes and other biomolecules. It can be also modified to present novel characteristics, creating innumerous applications, as for example in the food industry, as sweetener or thickening and gelling agent. Enzymatic conversion of starch to free glucose requires the concurrence of two enzymes: *α*-amylase, that yields malto-oligosaccharides and dextrins of varying chain length, and *α*-(1,4)-glucosidase (maltase), which hydrolyses terminal, non-reducing *α*-1,4-linked D-glucose residues with release of free D-glucose. These two enzymes can be replaced by amyloglucosidase (glucoamylase), a single enzyme able to break simultaneously the *α*-D-(1-4) and the *α*-D-(1-6), glycosidic bonds of both poly- and oligosaccharides. Efficient amylase-producing species include those bacteria of genus *Bacillus* (e.g. *B. licheniformis, B. subtilis, B. stearothermophilus, B. amyloliquefaciens*) and fungi of genus *Aspergillus* (e.g. *A. niger, A. oryzae, A. awamori, A. fumigatus*). Amylases makes up today up to 25% of the world enzyme market (personal communication, August, 2017), and are together with proteases, the most versatile enzymes in the industrial enzyme sector because of the abundance of substrates, raw materials and variety of applications as bakery goods, sugar products, biofuel industry, and many others.

Starch can be modified by chemical methods and an example are those termed "acid-thinned", normally used for food and beverage applications that involve an existing high starch content. Both *α*-1,4 and *α*-1,6 glucosidic linkages are moderately resistant to acid hydrolysis, with the amorphous regions of the granule more susceptible to chemical treatment than the crystalline regions. Acid modified starches are prepared industrially by treating the starch slurry (40%) with varying concentrations of mineral acids and hydrolysis time at temperatures below that of gelatinization (25–55°C) [61]. Acid treatment increases the gelatinization parameters (gelatinization temperature and enthalpy), reduces the molar mass and viscosity, increases the solubility of the granules, minimizes syneresis (separation of liquid from a gel caused by contraction), and causes gel thermo-reversibility when subjected to cooling after melting [61].

**Figure 2.** Effect of pH on the hydrolysis of cassava starch (30%) by phosphoric acid (a) and hydrochloric acid (b) at the temperature of 160°C (5 bar) during 10 min. Final concentrations of hydrolysis products and the final DE are shown. Key: (– x –) dextrose equivalents (DE), (■) glucose, (●) maltose, (□) maltotriose, (○) maltotetraose and higher.

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**Figure 3.** Example of sweeteners and GRASE status (generally regarded as safe and effective) of phosphoric and/or citric acids or their salts. (A) Cola soft drink containing HFCS or steviol glycosides and phosphoric acid; (B) fermented milk containing sweeteners from corn (high-fructose corn syrup - HFCS and/or modified corn starch) phosphoric and/or citric

acids or their salts (Source: Personal photo, 2015).

Acid Hydrochloric and sulfuric acid, more often the second, are the generally used mineral/ inorganic acids for starch hydrolysis, but they can present several problems. When using hydrochloric acid, in downstream step is necessary to desalinate the syrup using high cost ion exchange resins. Additionally, undesirable byproducts are produced even when syrups of an average dextrose equivalents are produced, because free glucose is converted to dehydration products such as hydroxymethylfurfural (HMF), levulinic and formic acids, which in turn can inhibit microbial growth if a subsequent fermentation step is required [62]. Furthermore, these mineral acids can easily produce toxic gases in the course of the process [63]. In the food and beverage industry, for example, another problem arises from the Maillard reactions between reducing sugars and R-NH2 groups from amino acids and proteins. This negative occurrence is designed as "mud" in the starch-processing factories of glucose-enriched syrups. Its worse properties are brown color and bitter taste [63].

The use of phosphoric acid instead of the stronger mentioned acids presents several advantages: safer handing because is a non-volatile acid, reduced byproducts formation and if the hydrolysate is to be used in a subsequent fermentation step, there is no need to remove or eliminate the phosphoric acid catalyst. Instead, neutralization with ammonia leads the formation of ammonium phosphate, a convenient supplement for growth as P and N-source. Taken together, these various considerations create the assumption of phosphoric acid as the preferred acid catalyst. From a strict biochemical stand point, let us to recall how much phosphoric acid is a "body friend" molecule: it is present in DNA, ATP, casein and phosphoricesters (Gluc- and Fruct-phosphates feeding the universal glycolytic pathway).

Our study comparing hydrolysis with phosphoric acid and hydrochloric acid on cassava starch paste (30%, w/v) has shown that at 160°C (*ca.* 6 atm), the desired dextrose equivalent (DE) was obtained with both acids: DE value of 85 at pH = 1.6 and pH = 1.8 using hydrochloric acid and a DE value of 83 at pH = 1.4 with phosphoric acid (**Figure 2**) [63]. Higher Sugar Versatility—Chemical and Bioprocessing of Many Phytobiomass Polysaccharides Using… http://dx.doi.org/10.5772/intechopen.75229 255

and fungi of genus *Aspergillus* (e.g. *A. niger, A. oryzae, A. awamori, A. fumigatus*). Amylases makes up today up to 25% of the world enzyme market (personal communication, August, 2017), and are together with proteases, the most versatile enzymes in the industrial enzyme sector because of the abundance of substrates, raw materials and variety of applications as

Starch can be modified by chemical methods and an example are those termed "acid-thinned", normally used for food and beverage applications that involve an existing high starch content. Both *α*-1,4 and *α*-1,6 glucosidic linkages are moderately resistant to acid hydrolysis, with the amorphous regions of the granule more susceptible to chemical treatment than the crystalline regions. Acid modified starches are prepared industrially by treating the starch slurry (40%) with varying concentrations of mineral acids and hydrolysis time at temperatures below that of gelatinization (25–55°C) [61]. Acid treatment increases the gelatinization parameters (gelatinization temperature and enthalpy), reduces the molar mass and viscosity, increases the solubility of the granules, minimizes syneresis (separation of liquid from a gel caused by contraction), and causes gel thermo-reversibility when subjected to cooling after

Acid Hydrochloric and sulfuric acid, more often the second, are the generally used mineral/ inorganic acids for starch hydrolysis, but they can present several problems. When using hydrochloric acid, in downstream step is necessary to desalinate the syrup using high cost ion exchange resins. Additionally, undesirable byproducts are produced even when syrups of an average dextrose equivalents are produced, because free glucose is converted to dehydration products such as hydroxymethylfurfural (HMF), levulinic and formic acids, which in turn can inhibit microbial growth if a subsequent fermentation step is required [62]. Furthermore, these mineral acids can easily produce toxic gases in the course of the process [63]. In the food and beverage industry, for example, another problem arises from the Maillard reactions between reducing sugars and R-NH2 groups from amino acids and proteins. This negative occurrence is designed as "mud" in the starch-processing factories of glucose-enriched syr-

The use of phosphoric acid instead of the stronger mentioned acids presents several advantages: safer handing because is a non-volatile acid, reduced byproducts formation and if the hydrolysate is to be used in a subsequent fermentation step, there is no need to remove or eliminate the phosphoric acid catalyst. Instead, neutralization with ammonia leads the formation of ammonium phosphate, a convenient supplement for growth as P and N-source. Taken together, these various considerations create the assumption of phosphoric acid as the preferred acid catalyst. From a strict biochemical stand point, let us to recall how much phosphoric acid is a "body friend" molecule: it is present in DNA, ATP, casein and phosphoric-

Our study comparing hydrolysis with phosphoric acid and hydrochloric acid on cassava starch paste (30%, w/v) has shown that at 160°C (*ca.* 6 atm), the desired dextrose equivalent (DE) was obtained with both acids: DE value of 85 at pH = 1.6 and pH = 1.8 using hydrochloric acid and a DE value of 83 at pH = 1.4 with phosphoric acid (**Figure 2**) [63]. Higher

esters (Gluc- and Fruct-phosphates feeding the universal glycolytic pathway).

bakery goods, sugar products, biofuel industry, and many others.

ups. Its worse properties are brown color and bitter taste [63].

melting [61].

254 Sugarcane - Technology and Research

**Figure 2.** Effect of pH on the hydrolysis of cassava starch (30%) by phosphoric acid (a) and hydrochloric acid (b) at the temperature of 160°C (5 bar) during 10 min. Final concentrations of hydrolysis products and the final DE are shown. Key: (– x –) dextrose equivalents (DE), (■) glucose, (●) maltose, (□) maltotriose, (○) maltotetraose and higher.

**Figure 3.** Example of sweeteners and GRASE status (generally regarded as safe and effective) of phosphoric and/or citric acids or their salts. (A) Cola soft drink containing HFCS or steviol glycosides and phosphoric acid; (B) fermented milk containing sweeteners from corn (high-fructose corn syrup - HFCS and/or modified corn starch) phosphoric and/or citric acids or their salts (Source: Personal photo, 2015).

temperatures and lower pH values led to higher concentrations of HMF and formic acid with both acids, but these quantities were always lower when hydrolysis was carried out with phosphoric acid (e.g. for HMF at pH = 1.5 and 152°C, 4 bar with a holding time of 5 min:

Our oPA-mediated cassava starch hydrolysate allowed biomass growth and astaxanthin production by the heterobasidiomycetous yeast *Xanthophyllomyces dendrorhous* (formerly: *Phaffia rhodozyma*) with parallel consumption of all maltosugars from G2 to G6 from an initial 64% of reducing sugars) reaching a maximum 3.34 mg/L of astaxanthin in a culture medium containing 6.5% w/v starch hydrolysate with supplementation (0.05 g/L yeast extract, and

phosphoric acid can be used as alternative catalyst to produce high DE syrups from cassava and other starch sources, residual phosphate being left in the final hydrolysate (after a light neutralization with ammonia or other alkalis) as a fermentation co-nutrient feedstock to

It may be remembered that sweetening of soft drinks and fermented milk (lacteous beverages and yogurts) can be attained with several alternatives of natural sugars or artificial sweeteners. Any of them are always accompanied with some phosphoric or citric acid and/ or their salts, as shown in the following illustration with two worldwide sold products

oPa-catalyzed partially starch hydrolysates (and even better if fructo-oligosaccharides, commonly known as FOS, the nutraceutical and anti-tumor oligosaccharides of inulin) along with the retained oPA catalyst within the hydrolysates may be a clever strategy to these product industrial formulas. Our group is waiting for the patent request PI 0703206-4 grant from the Brazilian INPI—National Institute for Industrial Property. Its claim includes the protection of the utilization of phosphoric and citric acids as mild catalysts for the production of FOS from *Dahlia* sp. tubers inulin. One example of such occurrence and some properties of inulin are

**Figure 6.** High performance liquid chromatography (HPLC) monitoring of the degree of polymerization (DP) of oPApartially hydrolyzed 10% inulin with pH 2.0 at 85°C for 5 min (bottom line) or 15 min (upper line). (Source: Authors lab).

PO4

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) [63].

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257

[63]. Results have shown that that diluted thermopressurized

0.185 mg/mL using HCl against 0.075 mg/mL with H3

NO3

25 to 50 mg/L of NH4

produce biomolecules [63].

(**Figure 3A** and **B**).

shown in the **Figures 4**–**7**.

FOS: fructo-oligosaccharides; RID: refractive index detector.

**Figure 4.** *Dahlia* sp. garden cultivation offers the most productive source for inulin which through a quick extraction of decorticated and sliced tubercles with pH 7 buffered hot water, followed by polymer retrogradation in a cold (ca. 8°C) environment. (Source: Prof. J. D. Fontana private auto-photo album).

**Figure 5.** 13C-Nuclear Magnetic Resonance (NMR) of purified *Dahlia* sp. tubercles inulin. The presence of minor spectroscopy signals – asterisk labels – correspond to the single glucopyranosyl of each whole inulin molecule, thus revealing that extraction and purification steps were carefully carried and preserving the polysaccharide native chemical structure. (Source: authors lab associate).

temperatures and lower pH values led to higher concentrations of HMF and formic acid with both acids, but these quantities were always lower when hydrolysis was carried out with phosphoric acid (e.g. for HMF at pH = 1.5 and 152°C, 4 bar with a holding time of 5 min: 0.185 mg/mL using HCl against 0.075 mg/mL with H3 PO4 ) [63].

Our oPA-mediated cassava starch hydrolysate allowed biomass growth and astaxanthin production by the heterobasidiomycetous yeast *Xanthophyllomyces dendrorhous* (formerly: *Phaffia rhodozyma*) with parallel consumption of all maltosugars from G2 to G6 from an initial 64% of reducing sugars) reaching a maximum 3.34 mg/L of astaxanthin in a culture medium containing 6.5% w/v starch hydrolysate with supplementation (0.05 g/L yeast extract, and 25 to 50 mg/L of NH4 NO3 [63]. Results have shown that that diluted thermopressurized phosphoric acid can be used as alternative catalyst to produce high DE syrups from cassava and other starch sources, residual phosphate being left in the final hydrolysate (after a light neutralization with ammonia or other alkalis) as a fermentation co-nutrient feedstock to produce biomolecules [63].

It may be remembered that sweetening of soft drinks and fermented milk (lacteous beverages and yogurts) can be attained with several alternatives of natural sugars or artificial sweeteners. Any of them are always accompanied with some phosphoric or citric acid and/ or their salts, as shown in the following illustration with two worldwide sold products (**Figure 3A** and **B**).

**Figure 4.** *Dahlia* sp. garden cultivation offers the most productive source for inulin which through a quick extraction of decorticated and sliced tubercles with pH 7 buffered hot water, followed by polymer retrogradation in a cold (ca. 8°C)

**Figure 5.** 13C-Nuclear Magnetic Resonance (NMR) of purified *Dahlia* sp. tubercles inulin. The presence of minor spectroscopy signals – asterisk labels – correspond to the single glucopyranosyl of each whole inulin molecule, thus revealing that extraction and purification steps were carefully carried and preserving the polysaccharide native chemical

environment. (Source: Prof. J. D. Fontana private auto-photo album).

256 Sugarcane - Technology and Research

structure. (Source: authors lab associate).

oPa-catalyzed partially starch hydrolysates (and even better if fructo-oligosaccharides, commonly known as FOS, the nutraceutical and anti-tumor oligosaccharides of inulin) along with the retained oPA catalyst within the hydrolysates may be a clever strategy to these product industrial formulas. Our group is waiting for the patent request PI 0703206-4 grant from the Brazilian INPI—National Institute for Industrial Property. Its claim includes the protection of the utilization of phosphoric and citric acids as mild catalysts for the production of FOS from *Dahlia* sp. tubers inulin. One example of such occurrence and some properties of inulin are shown in the **Figures 4**–**7**.

**Figure 6.** High performance liquid chromatography (HPLC) monitoring of the degree of polymerization (DP) of oPApartially hydrolyzed 10% inulin with pH 2.0 at 85°C for 5 min (bottom line) or 15 min (upper line). (Source: Authors lab). FOS: fructo-oligosaccharides; RID: refractive index detector.

**Figure 7.** Thin-layer chromatography (TLC) monitoring of inulin (poly-D-fructofuranose) by oPA-catalyzed partial or total hydrolysis along 15 min of incubation at 75°C: pH 4 (*left*), pH 3 (*cente*r) and pH 2 (*right*). Revelator: Hot 0.5% orcinol in MeOH:H2 SO4 9:1. The major spot at Rf = 0.8 is free fructose (F´). The multiband profile (*right*) are FOS (Fructo-oligoSaccharides) with degree of polymerization (DP) from 2 till 10. The two spots ahead fructose are HMF (HydroxyMethylFurufuraldeyde) and probably some DFA (DiFructose anhydride) due to acid reversion of free fructose.

**7. Conclusions**

processes.

**Acknowledgements**

Great potential is observed in the deconstruction of phytobiomass polysaccharides to its component sugars. Resulting monomers and/or oligomers can be used for the production of a plethora of products, with application to biofuels, food, fine chemicals and other industries. As mentioned before, due to the inherent characteristics of phosphoric acid, it can be used as an advantageous catalyst for the depolymerization of polysaccharides from a great variety of phytobiomass. Pretreatment technology using very diluted phosphoric acid, alone and under moderated thermopressurization for the bioprocessing of a sugarcane and other L(h)C substrates can possess important advantages over the use of mineral/inorganic acids, despite its relatively higher cost when compared to sulfuric acid, for example. We have shown the potential for using phosphoric acid hydrolysates to fermentation processes using different microorganisms, to the production of bioethanol, to increase nutritional value in animal feed, for starch modification, biomass growth and in the production of prebiotic/alternative sweetener (fructo-oligosaccharides). A continuous study in the use diluted phosphoric acid on different biomass could improve strategies that can be further used in industry and biorefinery

**Figure 8.** Thin-layer chromatography (TLC) of Nutraceutical oligosaccharides (NOs) arising from diluted thermopressurized oPA-catalyzed treatment of two microalgae and one cyanobacterium cell walls. Real effective pH 2.0 (after equilibration and complete wetting of each microorganism cell mass) and then a thermopressurization at 4.5 atm (156°C) till the peak condition for 2 minutes. Mobile phase: Acetonitrile:Isopropanol:Water (15:3:5). Chromogenic reagent: Orcinol in sulfuric acid. Standards: (Gal A) galacturonic acid, (Rha) rhamnose, (Man) mannose, (Ara) arabinose, (Gal) galactose, (Xyl) xylose, Rf = 0.48 - (Glu) glucose, Rf 0.38 - (Cb) cellobiose and (Mt) maltose, Rf = 0.29 - (Mtt) maltotriose, (XOS) xylo-oligosaccharides, (COS) cello-oligosaccharides, Rf = 0.83 - (HMF) hydroxymethylfurfural. (Source: Authors; picture abstracted from Bruna Leal master dissertation, supervised by Prof. Marcelo R. Prado and Adéia Grzybowski, 2015).

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The authors are grateful to the main Brazilian Agencies financial support: CNPq—National Council for S&T Development, CAPES—Coordination for the Improvement of Persons of
